CN101690809A - Propolis-astragalus polysaccharides compound nanometer emulsion adjuvant and preparation method and application thereof - Google Patents
Propolis-astragalus polysaccharides compound nanometer emulsion adjuvant and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a propolis-astragalus polysaccharides compound nanometer emulsion adjuvant which comprises the following components by weight percent: 0.01%-6% of propolis, 0.01%-14% of astragalus polysaccharides, 16.6%-54.0% of surfactant, 6.3%-30.5% of cosurfactant, 5.1%-25.2% of oil and 34.0%-60.0% of distilled water. The sum of the weight percentages of the components is 100%. The compound nanometer emulsion adjuvant is brown-yellow transparent oil-in-water nanometer emulsion with the grain size of 1-100nm. The compound nanometer emulsion adjuvant can be directly used to prepare vaccine along with various inactivated antigens and the mass production can be realized without any special emulsifying device. Meanwhile, the compound adjuvant has the advantage of good stability, high safety, low viscosity and the like.
Description
Technical field
The present invention relates to a kind of adjuvant of new generation vaccine, be specifically related to the preparation method of a kind of propolis and astragalus polysaccharides compound nanometer emulsion adjuvant and the application in production of vaccine thereof.
Technical background
Because there are many safety issues in live vaccine, inactivated vaccine and various new generation vaccine more and more are subject to people's attention.But characteristics such as inactivated vaccine and various new generation vaccine ubiquity immunogenicity differences can not play good clinical protection effect.In order to strengthen the immune effect of vaccine, must cooperate adjuvant to strengthen its immunogenicity, the focus that therefore, develop in recent years safely, stable immunological adjuvant has become people's concern.At present, the fatal shortcoming that inactivated vaccine and gene engineering vaccine exist is can't the inducing T cell immunoreation, but in inducing more effective antiviral immunity reaction, the participation of T cell is absolutely necessary, and adjuvant can be assisted antigen induction T cellular immunization.Simultaneously more need adjuvant to antigenic protection be directed to the effect organ and tissue for mucosal immunity approach.
Summary of the invention
At above-mentioned problems of the prior art and defective, the object of the present invention is to provide a kind of propolis and astragalus polysaccharides novel compound nano emulsion adjuvant, this compound adjuvant can significantly improve the immunogenicity of inactivation antigen, overcome shortcomings such as traditional adjuvant instability, viscosity height, tissue reaction be big simultaneously, and preparation technology is simple, low production cost is joined Seedling conveniently, does not need special installation promptly to can be mass-produced.
The technical scheme that realizes the foregoing invention purpose is a kind of propolis and astragalus polysaccharides compound nanometer emulsion preparation, it is characterized in that it is to be made by following weight percentages:
Propolis 0.01%～6%, astragalus polysaccharides 0.01%～14%, surfactant 16.6%～54.0%, cosurfactant 6.3%～30.5%, oil 5.1%～25.2%, distilled water 34.0%～60.0%, the total weight percent of above-mentioned raw materials is 100%.
The optimum ratio of propolis of the present invention and astragalus polysaccharides compound nanometer emulsion preparation is: propolis 0.10%～5%, astragalus polysaccharides 0.10%～12%, surfactant 20.0%～50.0%, cosurfactant 5.0%～30.0%, oil 4.5%～25.0%, distilled water 40.0%～55.0%.
Described surfactant is RH-40, and cosurfactant is a dehydrated alcohol, and oil is ethyl acetate.
The propolis that the present invention selects for use (Propolis) is a kind of natural active matter that contains the various active composition, and wherein enzyme, alcohols, lipid and acid have immunological effect widely, has been widely used in the prevention of poultry infectious disease.Propolis has the various biological function, as antibiotic, antiinflammatory, antioxidation and immunomodulating.Multinomial research data shows that the propolis vaccine can start the immune defence system of body comprehensively, stimulates body cell immunity, humoral immunization, erythrocyte immune system and macrophage complement system all to produce immunne response.Propolis as the research of adjuvant report show the propolis Seedling have protective rate height, duration of immunity long, stress be little, advantage such as have no side effect.
The astragalus polysaccharides that the present invention selects for use (Astragalus Polysaccharides APS) has multiple biological activity as polysaccharose substance, at present in veterinary clinic as immunomodulator and vaccine adjuvant extensive use.Multinomial research data shows, the specific immunity that astragalus polysaccharides can not only enhancing body, and nonspecific immunity that can enhancing body both can strengthen the immunologic function of normal body, again the unusual immunologic function of scalable body.
Therefore, the present invention prepares propolis and astragalus polysaccharides compound nanometer emulsion adjuvant by the nano-emulsion medicine-carried system, and purpose is to improve the immunogenicity of vaccine, lays the foundation for this invention can be applied to actual production.
The preparation method of above-mentioned compound bee-glue and astragalus polysaccharides nano-emulsion preparation is provided in a further object of the invention, may further comprise the steps:
1) take by weighing propolis, astragalus polysaccharides, surfactant, cosurfactant, oil, distilled water, standby;
2) propolis is dissolved in the cosurfactant fully, and then adds oil, add the surfactant mixing at last, standby;
3) astragalus polysaccharides is dissolved in distilled water;
4) slowly drip the astragalus polysaccharides aqueous solution of step 3) preparation under the room temperature to step 2) in the solution of preparation, constantly stir simultaneously, until the system that forms homogeneous transparent, promptly.
Provide above-mentioned compound bee-glue and astragalus polysaccharides nano-emulsion preparation to subtract application in the poultry inactivated vaccines such as syndrome, the blue ear of pig preparation bird flu, newcastle, chicken in a further object of the invention.
Among the present invention, because propolis is insoluble in water, astragalus polysaccharides is soluble in water, therefore the aqueous solution of astragalus polysaccharides as aqueous portion, propolis as the oil phase part.Adopt the pseudo-ternary phase diagram method to filter out the optimum formula of preparation compound bee-glue nano-emulsion.Surfactant is RH-40, and cosurfactant is a dehydrated alcohol, and oil is ethyl acetate.When surfactant and cosurfactant mass ratio are 2: 1, surfactant and cosurfactant sum are the nano-emulsion district maximum that formed in 9: 1～6: 4 o'clock with the mass ratio of oil, and the most stable, drug loading is the highest.RH-40 toxicity used in the prescription is less relatively, is not subject to the influence of electrolyte, inorganic salts and soda acid; The nano-emulsion that anhydrous cosurfactant forms is not only stable, and can be used as the propolis solvent, has improved the content of propolis in this compound nanometer emulsion.
Utilize speed that oil-soluble dyes tonyred and water-soluble dye methylene blue spread in propolis and astragalus polysaccharides compound nanometer emulsion to judge the type of nano-emulsion, find that methylene blue solution has diffusion in nano-emulsion, tonyred solution is then not diffusion in nano-emulsion, shows that prepared compound nanometer emulsion is oil-in-water type (O/W).
With prepared propolis and the centrifugal 20min of astragalus polysaccharides compound nanometer emulsion 20000r/min, reserved sample observing is 3 months under 4 ℃, 25 ℃, 40 ℃ conditions, observes every the 10d sampling, does not all find breakdown of emulsion, flocculation and lamination, and the outward appearance no change.
Propolis of the present invention and astragalus polysaccharides compound nanometer emulsion appearance transparent, particle diameter are between 1～100nm, and its viscosity can add the distilled water adjustment of arbitrary proportion as required, conveniently joins Seedling, and two kinds of raw material adjuvant propolis and astragalus polysaccharides character all do not change.
Propolis of the present invention and astragalus polysaccharides compound nanometer emulsion are safe, and through acute oral toxicity test, local excitation test and pyrogenic test, animal subject is in a good state of health, and untoward reaction all do not occur.
Compound bee-glue of the present invention and astragalus polysaccharides nano emulsion adjuvant can be used for joining Seedling with multiple inactivated vaccine and new generation vaccine, and a step finishes, and need not emulsifying.
Compared with prior art, compound nanometer emulsion of the present invention has the following advantages:
1) compound nanometer emulsion adjuvant of the present invention has the stability of pharmaceutical preparation excellence, and high speed centrifugation still keeps the clear state, phenomenons such as layering, breakdown of emulsion do not occur.
2) compound nanometer emulsion adjuvant of the present invention is safe, and to the live body nonhazardous, the injection site does not have phenomenons such as granuloma and inflammatory reaction, does not occur the side reaction of vaccine after the injection.
3) compound nanometer emulsion adjuvant viscosity of the present invention is low, makes things convenient for drug administration by injection.
4) to join Seedling technology simple for compound nanometer emulsion adjuvant of the present invention, do not need large-scale emulsifying device promptly to be produced on a large scale, and reduced production cost.
Description of drawings
The transmission electron microscope photo of Fig. 1 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant.
The particle size distribution figure of Fig. 2 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant.
The specific embodiment
Following examples mainly are to be used to further specify the present invention, rather than limit the scope of the invention.
The preparation of embodiment 1 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Preparation prescription is as follows:
Propolis 1.0g
RH-40???????????????????????????33.0g
Dehydrated alcohol 16.5g
Ethyl acetate 5.5g
Astragalus polysaccharides 1.0g
Distilled water 42.5g
The concrete operations step is:
1) it is standby to take by weighing propolis, astragalus polysaccharides, surfactant, cosurfactant, oil, distilled water respectively in the prescription ratio;
2) propolis with recipe quantity is dissolved in cosurfactant (cosurfactant can be used as solvent here) fully, adds oil phase again, adds the surfactant mixing at last, and is standby.
3) by recipe quantity astragalus polysaccharides is dissolved in distilled water;
4) slowly drip above-mentioned distilled water under the room temperature in system, constantly stir simultaneously, the system until forming homogeneous transparent promptly makes propolis and the astragalus polysaccharides nano-emulsion preparation of particle diameter in 1～100nm, appearance transparent.
Embodiment 2 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 0.01g
RH-40???????????????????????????33.0g
Dehydrated alcohol 16.5g
Ethyl acetate 5.5g
Astragalus polysaccharides 0.1g
Distilled water 37.5g
Embodiment 3 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 0.1g
RH-40???????????????????????33.0g
Dehydrated alcohol 16.5g
Ethyl acetate 5.5g
Astragalus polysaccharides 14.0g
Distilled water 34.5g
Embodiment 4 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 0.50g
RH-40???????????????????????31.4g
Dehydrated alcohol 15.7g
Ethyl acetate 8.3g
Astragalus polysaccharides 1.0g
Distilled water 38.6g
Propolis 1.0g
RH-40???????????????????????29.0g
Dehydrated alcohol 14.5g
Ethyl acetate 11.5g
Astragalus polysaccharides 5.0g
Distilled water 40.0g
Embodiment 6 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 1.50g
RH-40???????????????????????????27.6g
Dehydrated alcohol 13.8g
Ethyl acetate 13.8g
Astragalus polysaccharides 0.5g
Distilled water 39.8g
Embodiment 7 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 3.0g
RH-40???????????????????????????25.0g
Dehydrated alcohol 12.5g
Ethyl acetate 16.5g
Astragalus polysaccharides 0.01g
Distilled water 41.5g
Embodiment 8 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 2.0g
RH-40???????????????????????????23.0g
Dehydrated alcohol 11.5g
Ethyl acetate 22.0g
Astragalus polysaccharides 1.0g
Distilled water 55.0g
The preparation of embodiment 9 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 0.01g
Astragalus polysaccharides 0.01g
RH-40???????????????????????23.0g
Dehydrated alcohol 11.5g
Ethyl acetate 22.0g
Distilled water 43.48g
Propolis 2g
Astragalus polysaccharides 5g
RH-40???????????????????????29.0g
Dehydrated alcohol 14.5g
Ethyl acetate 11.5g
Distilled water 38g
Embodiment 11 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 3g
Astragalus polysaccharides 7.0g
RH-40????????????????????????31.4g
Dehydrated alcohol 15.7g
Ethyl acetate 8.3g
Distilled water 60.g
Embodiment 12 propolis and astragalus polysaccharides compound nanometer emulsion adjuvant
Propolis 6g
Astragalus polysaccharides 14g
RH-40????????????????????????33.0g
Dehydrated alcohol 16.5g
Ethyl acetate 5.5g
Distilled water 50g
The preparation of embodiment 13 newcastle disease compound bee-glue nano emulsion adjuvant inactivated vaccines
Under gnotobasis, room temperature is lower than 30 ℃, with the composite propolis nano emulsion adjuvant of preparation, directly mixes with the newcastle inactivation antigen according to the ratio of adjuvant consumption 30～60% (above is volume fraction), fully shakes up; Also can adopt magnetic stirring apparatus 300r/min～1000r/min to stir 3～5 minutes, can make the newcastle disease compound bee-glue nano emulsion adjuvant inactivated vaccine of outward appearance clear.
The preparation of the blue ear compound bee-glue of embodiment 14 pigs nano emulsion adjuvant inactivated vaccine
Under gnotobasis, room temperature is lower than 30 ℃, with the composite propolis nano emulsion adjuvant of preparation, directly mixes with the blue ear inactivation antigen of pig according to the ratio of adjuvant consumption 30～60% (above is volume fraction), fully shakes up; Also can adopt magnetic stirring apparatus 300r/min～1000r/min to stir 3～5 minutes, can make the blue ear compound bee-glue of the pig nano emulsion adjuvant inactivated vaccine of outward appearance clear.
The concrete using method of compound nanometer emulsion adjuvant of the present invention:
Before joining Seedling with inactivation antigen, the nanometer adjuvant is suitably diluted, make propolis content 20mg/mL in its nano-emulsion, astragalus polysaccharides content 10mg/mL with water for injection or distilled water.Ratio according to adjuvant consumption 30～60% (more than be volume fraction) is directly mixed with various inactivation antigens, fully shakes up; Also can adopt magnetic stirring apparatus 300r/min～1000r/min to stir 3～5 minutes, can make compound bee-glue nano emulsion adjuvant vaccine.
Test example 1 propolis and astragalus polysaccharides compound nanometer emulsion stability of formulation are investigated
1) centrifugal acceleration test is to the nano-emulsion stability influence
Get the nanoemulsions of the present invention of preparation, centrifugal with 20000r/min, to observe the nanoemulsions outward appearance behind the 10min and still keep clear, the profit lamination does not appear.
2) light stability test
Nano-emulsion of the present invention is packed in the vial in right amount, and room temperature is placed under (4500 ± 500) the lx illumination condition of sealing back, respectively at 0d, 5d, the 10d observation of taking a sample.The result shows that emulsion keeps the clear outward appearance, does not see phenomenons such as layering and breakdown of emulsion.
3) reserved sample observing test
Emulsion of the present invention is sub-packed in the vial, and placing respectively after the sealing keeps sample under 4 ℃, 25 ℃, the 40 ℃ conditions investigates 6 months, observes every the 10d sampling.The result shows that emulsion all keeps the clear outward appearance under these four kinds of temperature conditions, do not see phenomenons such as layering and breakdown of emulsion.
The safety of test example 2 propolis and astragalus polysaccharides compound nanometer emulsion preparation is investigated
1 experimental technique
1.1 acute oral toxicity test
Select 10 kunming mices for use, male and female half and half, overnight fasting, not water restriction before the test.Get P-APS-NE 2g, use the 20mL physiological saline solution, be mixed with the 100mg/mL aqueous solution.By every mice maximum dosage-feeding 0.1g, promptly every mice administration 1mL divides perfusion in two hours.Irritated the back and continued fasting 3h～4h, during can suitably give a small amount of drinking-water according to the mice reaction.14d regularly observes 10 mices after irritating stomach, the itemized record reaction.
1.2 acute eye irritation test
Select 3 adult white rabbit for use, to two eye examinations of rabbit, what eye irritation symptom, cornea defective and conjunctival damage were arranged can not be used for test before the experiment.Draw back the palpebra inferior of rabbit left side eyes during test gently, 0.1mL splashes into left eye with trial target, makes the passive closed 2s of palpebra inferior, and the right side eyes splash into distilled water and do contrast.After the administration 1,24,48 and 72h check the reaction and the record of eyes.Carry out scoring of eye irritant reaction and classification according to table 1 and table 2.
The standards of grading of table 1 eye irritant reaction
The classification of table 2 eye irritant reaction
1.3 muscle irritation test
Get 3 of 3 batches of test samples difference intramuscular injection healthy rabbits, injection compound bee-glue nano emulsion adjuvant 1mL in the one rear flank limb quadriceps femoris, opposite side hind leg corresponding position injecting normal saline 1mL behind the 48h puts to death rabbit the variation of anatomic observation injection site muscular tissue in contrast.
1.4 pyrogen testing
Get 3 of suitable rabbit (doe should not have pregnant), after measuring its normal temperature, in the 15min, slowly inject the P-APS-NE injection 1mL/kg that is preheating to 38 ℃ of prescribed dose, survey body temperature 1 time every 60min by preceding method then, totally 4 times from auricular vein.Once deduct the rising number of degrees that normal body temperature is this rabbit body temperature with the highest in 4 body temperature.In 3 rabbit, if fervescence all below 0.6 ℃, and the fervescence sum of 3 rabbit should think that below 1.4 ℃ test sample is up to specification.
2 experimental results
2.1 acute oral toxicity
In 14d behind the maximal dose filling stomach, death all appears in 10 mices, intoxicating phenomenon also do not occur.Irritated stomach mice food-intake the same day and descend irritating stomach, 2d recovers, and viewing duration mice food-intake, all normal by hair, body weight with blank mice zero difference, shows this compound adjuvant per os LD50＞5000mg/kg, and true border is nontoxic.
2.2 tame rabbit acute eye stimulating
3 rabbit in the 72h, relatively do not have any variation with the own control eyes after eyes splash into compound nanometer emulsion, total mark is 0 fen, show that compound bee-glue astragalus polysaccharides nano-emulsion does not have eye irritation.
2.3 rabbit muscular irritation
Cuing open inspection position test group and matched group does not have obvious difference, phenomenons such as the hyperemia of local nothing, edema, degeneration or necrosis.Show that compound bee-glue astragalus polysaccharides nano-emulsion does not have muscle irritation.
2.4 pyrogen testing
Table-3 pyrogen testing results
Remarks: check data 08.7.12,25 ℃ of room temperatures.
After injecting P-APS-NE as calculated, experimental rabbit body temperature changes little, and every variation all is no more than 0.6 ℃, and summation also is no more than 1.4 ℃, so this test sample meets the inspection requirements of pyrogen quality.
The immune contrast test of test example 3 compound bee-glue nano emulsion adjuvants and white-oil adjuvant
1 materials and methods
1.1 antigen: newcastle inactivation antigen
1.214 test chicken in age (non-immune egg cock)
1.3 the test group experiment is made as blank group, white-oil adjuvant vaccine group, compound bee-glue nano emulsion adjuvant vaccine group.Wherein the antigen of white oil vaccine group and propolis vaccine group is identical with antigenic content, and adjuvant and antigenic volume ratio are 2: 1.
2 steps
2.1 the mensuration of serum antibody titer: every group of 20 chickens, every immune 0.5mL, chest muscle injection.Detect serum HI antibody titer respectively at 25 ages in days, 35 ages in days, 45 ages in days.Observe the injection site local response simultaneously.
2.2 before the immunity and immunity back 45 ages in days are measured chicken group's average weight gain, observe the influence of immunity to chicken group weightening finish.
3 results and analysis
The result shows, white-oil adjuvant and propolis adjuvant all can effectively promote the generation of immune chicken antibody, improve the humoral immunity of organism level, and compound bee-glue nano emulsion adjuvant immune effect of vaccine is better than the white-oil adjuvant vaccine, the immune side reaction that causes simultaneously is little, and concrete outcome sees the following form-4.
Table-4HI TPPA result (Log2)
| Grouping | 25 ages in days | 35 ages in days | 45 ages in days | Local response |
| Blank group white-oil adjuvant group | ??4.6??7.3 | ??4.2??6.8 | ??3.8??6.1 | Absorb good NIP and react slight inflammatory reaction |
| Compound bee-glue adjuvant group | ??8.2 | ??7.6 | ??7.1 | Absorb good NIP reaction |
Measure the influence of different adjuvants to the chicken weightening finish before and after the immunity, compound bee-glue adjuvant group can significantly promote the growth of chicken, and its average weight gain is greater than the white-oil adjuvant group, and concrete outcome sees Table-5.
Table-5 different Adjuvanted vaccines are to the influence of chicken weightening finish
| Group | Average weight (g) before the immunity | Immunity back 45 average weights in age (g) | Average weight gain (g) |
| Blank group white-oil adjuvant group | ?84?82 | ?896?883 | ?812?801 |
| Compound bee-glue adjuvant group | ?82 | ?976 | ?894 |
Claims (8)
1. propolis and astragalus polysaccharides compound nanometer emulsion adjuvant is characterized in that this nano-emulsion is an oil-in-water type, and its particle diameter is made by following weight percentages between 1～100nm:
Propolis 0.01%～6%
Astragalus polysaccharides 0.01%～14%
Surfactant 16.6%～54.0%
Cosurfactant 6.3%～30.5%
Oil 5.1%～25.2%
Distilled water 34.0%～60.0%
The total weight percent of above-mentioned raw materials is 100%;
Described surfactant is polyoxyethylene ether (a 40) castor oil hydrogenated (RH-40);
Described cosurfactant is a dehydrated alcohol;
Described oil is ethyl acetate.
2. propolis according to claim 1 and astragalus polysaccharides compound nanometer emulsion adjuvant is characterized in that, are made by following weight percentages:
Propolis 0.10%～5%
Astragalus polysaccharides 0.10%～12%
Surfactant 20.0%～50.0%
Cosurfactant 5.0%～30.0%
Oil 4.5%～25.0%
Distilled water 40.0%～55.0%
The total weight percent of above-mentioned raw materials is 100%.
3. claim 1 or 2 described compound nanometer emulsion adjuvants is characterized in that described surfactant and cosurfactant mass ratio are 2: 1.
4. prepare the method for described propolis of claim 1 and astragalus polysaccharides compound nanometer emulsion adjuvant, it is characterized in that, specifically may further comprise the steps:
1) take by weighing propolis, astragalus polysaccharides, surfactant, cosurfactant, oil, distilled water, standby;
2) propolis is dissolved in the cosurfactant fully, and then adds oil, add the surfactant mixing at last, standby;
3) astragalus polysaccharides is dissolved in distilled water;
4) slowly drip the astragalus polysaccharides aqueous solution of step 3) preparation under the room temperature to step 2) in the solution of preparation, constantly stir simultaneously, until the system that forms homogeneous transparent, promptly.
5. described propolis of claim 1 and the astragalus polysaccharides compound nanometer emulsion adjuvant application in the preparation inactivated avian influenza vaccine.
6. described propolis of claim 1 and the astragalus polysaccharides compound nanometer emulsion adjuvant application in preparation newcastle inactivated vaccine.
7. described propolis of claim 1 and astragalus polysaccharides compound nanometer emulsion adjuvant subtract application in the syndrome deactivation vaccine the preparation chicken.
8. described propolis of claim 1 and the astragalus polysaccharides compound nanometer emulsion adjuvant application in the blue ear inactivated vaccine of preparation pig.
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| CN107335055B (en) * | 2017-07-21 | 2020-05-01 | 商丘美兰生物工程有限公司 | Veterinary traditional Chinese medicine immunopotentiator and application thereof |
| CN108991453A (en) * | 2018-08-06 | 2018-12-14 | 重庆市畜牧科学院 | A kind of preparation method and products thereof of compound nano-propolis |
| CN110013551A (en) * | 2019-05-21 | 2019-07-16 | 中国水产科学研究院珠江水产研究所 | A kind of fishing immersion immunity composite adjuvant and the preparation method and application thereof |
| CN113855797A (en) * | 2021-11-19 | 2021-12-31 | 安徽科技学院 | Diluent of veterinary immunity vaccine and its preparing method |
| CN113855797B (en) * | 2021-11-19 | 2023-11-10 | 安徽科技学院 | Diluent for veterinary immune vaccine and preparation method thereof |
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